Tuberculosis (TB) is a chronic infectious disease caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb). The World Health Organization estimates that at least 2 million people die every year because of TB, and that one-third of the living world population is infected with Mtb. The recent emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug resistant TB (XDR-TB) strains, and the infection of AIDS patients by Mtb, present more challenges in the management and treatment of the disease. Identification of new drug targets and development vaccines are the immediate priorities in TB research. Attenuated Mycobacterium bovis BCG has long been used as a vaccine against TB, but its efficacy is variable for different populations. Now, however, several new and highly immunogenic antigenic peptides/proteins have been identified from M. tuberculosis. The secreted antigenic proteins Ag85B and ESAT- 6, when used together, can elicit protective immune responses in mice, guinea pigs and nonhuman primates. This vaccine provides efficacy equivalent to that of BCG, so there is a potential for this to be a prophylactic vaccine replacement for BCG, and a booster vaccine for those who had BCG vaccine earlier but with no protective immune response. However, these protein antigens present several logistical problems. First, the proteins must be overexpressed and purified in large quantities from bacterial vectors, making them expensive to produce. Second, the vaccine formulation requires storage in freezers. Finally, the vaccine immunogens must be injected intramuscularly, by medical personnel, for optimum effect. Alternate strategies are required for better use of this vaccine. We plan to engineer recombinant Bacillus subtilis spores that express Ag85B and ESAT-6, to be used as a particulate vaccine to TB. Bacillus spores are dormant and rugged, and maintain their integrity under extreme conditions, including low pH, desiccation, UV irradiation and temperatures of up to 90oC. Interestingly, Bacillus spores are harmless to humans; in fact, they are sold in grocery stores and are being consumed in a number of novel foods as probiotics. Bacillus spores do not require special storage, and can be orally and nasally administered. Using plasmid vectors, we first will clone the genes encoding the Ag85B and ESAT-6 proteins downstream of the cotC gene, which encodes spore coat protein C, and the rrno gene of B. subtilis. We will then integrate these constructs into the chromosome of B. subtilis through homologous recombination. Strains carrying this fusion construct will be caused to produce spores by nutritional deprivation. We will then test the spores for their ability to eliit anti-Mtb cellular immune responses, after intranasal or oral immunization of the mice. Finally, we will test whether mice with these immunization groups are protected against challenge doses of live M. tuberculosis H37Rv. PUBLIC HEALTH RELEVANCE: Tuberculosis (TB) is a serious disease that kills approximately two million people every year throughout the world. Attenuated Mycobacterium bovis BCG is currently used as a vaccine against TB, but its efficacy varies with different populations. Recent technological advances have allowed identification of new and highly immunogenic peptides/proteins from M. tuberculosis. However, several logistical problems must be overcome for application of these peptides as vaccine to humans. We propose to genetically engineer the bacterium Bacillus subtilis to express these proteins on its spores, so that the recombinant spores can be used as heat- stable, easily stored and easily administered mucosal vaccine against tuberculosis.